L-683,590 microbial transformation product

ABSTRACT

Described is a new L-679,934 (FK-506) antagonist, L-686,292, a C-15, C-31 bisdemethylated, derivative of L-683,590, produced under fermentation conditions utilizing the microorganism, Actinoplanacete sp. (Merck Culture Collection MA 6559) ATCC No. 53771. The macrolide reverses the immunosuppressant action of L-679,934 (FK-506), and can be used diagnostically as a tool to determine the presence of FK-506 macrolide type immunosuppressants in natural product broths, distinguishing such immunosuppressants from other chemical families such as the cycloporins.

This is a continuation of application Ser. No. 07/544,802, field Jun.25, 1990 now abandoned, which is a continuation of application Ser. No.07/366,096, filed Jun. 13, 1989 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a new L-679,934 immunosuppressant antagonist,L-686,292, and a fermentation process for its production utilizing themicroorganism Actinoplanacete sp. (MA 6559), ATCC No. 3771. The processinvolves culturing the microorganism and L-683,590 under conditionswhich effects C-15, C-31 bisdemethylation of L-683,590.

2. Brief Description of Disclosures in the Art

In 1983, the US FDA licensed cyclosporin, and extremely effectiveanti-rejection drug that revolutionized the field of organ transplantsurgery. The drug acts by inhibiting the body's immune system frommobilizing its vast arsenal of natural protecting agents to reject thetransplant's foreign protein.

As effective as the drug is in fighting transplantation rejection, itsuffers drawbacks in causing kidney failure, liver damage and ulcerswhich in many cases can be very severe.

EPO Publication No. 0184162 to Fujisawa, hereby incorporated byreference, describes a new macrolide immunosuppressant FK-506 which isreputed to be 100 times more effective than cyclosporin. The macrolideis produced by fermentation of a particular is the closely relatedmacrolide immunosuppressant FK-520, produced by S. hygroscopcus subsp.yakushimaensis.

U.S. Pat. No. 3,244,592 to T. Arai describes the culturing ofStreptomyces hygroscopicus var. ascomyceticus to produce the antifungal"ascomycin".

There is, however, no description in the literature of the production ofany L-679,934 antagonist which will substantially reverse theimmunosuppressant effects of L-679,934, and be useful in situationsrequiring the determination of FK-506 macrolide type activity as opposedto other chemical families such as the cyclosporins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an ¹ H nuclear magnetic resonance (NMR) spectrum laken at 400MHz to L-686,292 in CDCl₃ and illustrating the assigned molecularstructure for L-686,292.

FIG. 2 is an ¹ H NMR spectrum taken at 400 MHz of L-683,590(immunomycin) in CDCl₃.

SUMMARY OF THE INVENTION

It has been found that a new L-679,934 antagonist, L-686,292, can beobtained by the fermentation of the microorganism Actinoolanacete so.(MA 6559), ATCC No. 53771, in the presence of the macrolideimmunosuppressant L-683,590, under submerged aerobic conditions in anaqueous carbohydrate medium, containing a nitrogen nutrient, saidconditions being conducted at a pH of about 7 for a sufficient time toselectively C-15, C-31 bisdemethylate L-683,590, i.e., remove two methylradicals from two different methoxyl groups. L-686,292 is a minorfermentation product found in the broth along with L-687,795, the C-13,C-15, C-31 trisdemethylated analog of L-683,590, disclosed in copendingSer. No. 348,243 (Case 17911), filed May 5, 1989, having the sameassignee, and hereby incorporated by reference. Major components in thebroth include L-683,756, a C-13, C-31 bisdemethylated ring rearrangedderivative of L-683,590 as described in Ser. No. 297,630 (Case 17832),filed Jan. 13, 1989, having the same inventorship and assignee, andL-683,742, the monodemethylated analog of L-683,590, as described inSer. No. 213,025 (Case 17767) filed Jun. 29, 1988, having the sameassignee, and both hereby incorporated by reference for this particularpurpose.

The resultant L-686,292 exhibits L-679,934 antagonist activity, i.e.,reversal of inhibition of T-cell activation caused by L-679,934, asdemonstrated by the calcium ionophore (ionomycin) plus phorbol myristateacetate (PMA) induced T-cell stimulation assay, also referred to hereinas the "T-cell proliferation assay". The principle of this assay is tomeasure the proliferation of mouse T lymphocytes stimulated with thecombination of ionomycin plus PMA. L-679,934 inhibits T-cellproliferation in this assay, as indicated by reduced tritiated thymidineuptake. This inhibition of T-cell proliferation can be reversed with acompound, e.g. L-686,292, that binds to the cytosolic binding protein ormolecule which mediates inhibition by L-679,934. In this case,competitive binding to the target molecule by the antagonist L-686,292does not mediate inhibition of T-cell proliferation. This newlydiscovered protein is disclosed in Ser. No. 300,659 (Case 17866) filedon Jan. 19, 1989, and hereby incorporated by reference for thisparticular purpose.

In accordance with this invention, there is provided an L-679,934antagonist, identified as L-686,292, produced by culturing a strain ofActinoplanacete sp. together with L-683,590 under submerged aerobicfermentation conditions in an aqueous carbohydrate medium, containing anitrogen nutrient, for a sufficient time to produce product L-686,292.

The new antagonist, L-686,292, exhibits reversal of L-679,934 mediatedinhibition of T-cell activation as measured in the T-cell proliferationassay, exhibits a proton nuclear magnetic resonance spectrum asidentified in FIG. 1, and a molecular weight of 763 as determined byelectron impact mass spectrometry.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention involves the fermentation of Actinoplanacete sp.MA 6559, together with L-683,590 to produce L-686,292. The microorganismis currently on restricted deposit with the American Type CultureCollection, 12301 Parklawn Drive in Rockville, Md. as ATCC No. 53771,and in the Merck Culture Collection in Rahway, N.J. as MA 6559. Thephysical characteristics and taxonomy, including morphological,cultural, biological and physiological characteristics are brieflydescribed hereinbelow.

On the basis of the taxonomic analysis performed thus far, the culturehas tentatively been assigned in the order Actinomycetales and in thefamily Actinoplanacea. Further taxonomic characteristics are beingexamined to place this organism conclusively within a genus and species.

This culture grows well on routine media including trypticase soy agar(28° and 37° C.), yeast malt extract agar, glycerol asparagine agar,inorganic salt starch agar, oatmeal agar, Czapek Dox, Czapek solutionagar and peptone agar, and Bennett's agar, all at 28° C.

Morphology--This culture grows as a branched filamentous mycelium with adiameter of 0.2-0.4 microns. Colonies are opaque, raised, and erose.Colony texture is rubbery on yeast malt extract agar but tends to bebutyrous on other media where significant fragmentation of the myceliumis observed. The colony surface tends to be powdery in appearance. Nodiffusable pigments were observed.

Sporgangia--are predominantly spherical and range in size from 4-25microns in diameter. Sporangia are generally visible by 21 days and tendto coalesce on glycerol asparagine agar. Spores are rod shaped withblunt ends (0.76×1.9 microns), non-motile and occur in long, unbranchedchains of up to 150 microns in length.

Cultural characteristics of MA 6559 Yeast Extract-Malt Extract Agar (ISPMedium 2)

Vegetative mycelium is hyaline to yellow, aerial mycelium develops in24-72 h and is buff to rose-pink and powdery in appearance. The reverseside is tan to reddish brown.

Oatmeal Agar (ISP Medium 3)

Vegetative mycelium is hyaline to yellow, the reverse side is hyaline totan. Aerial growth is white to light rose-beige and powdery inappearance.

Inorganic Salts-Starch Agar (ISP Medium 4)

Light growth, scant aerial mycelium. Vegetative growth is hyaline andhighly fragmented. Clearing of starch occurs at periphery of coloniesnoted by 7 d.

Glycerol Asparagine Agar (ISP Medium 5)

Vegetative growth is hyaline to yellow, the reverse side is hyaline tocinnamon brown. Aerial mycelium is powdery and white to rose-pink.

Peptide-Iron-Yeast Extract Agar (ISP Medium 6)

Vegetative growth is tan. No aerial growth observed, no melanoidpigments produced.

Tyrosine Agar (ISP Medium 7)

Vegetative growth is tan becoming deep purple as culture ages. Aerialmycelium is velvety to grayed rose-beige.

Czapek-Dox Agar

Vegetative growth is tan with a pink tone as the culture ages. Aerialmycelia are short and matted with a moist appearance.

The present invention process can be practiced with any"L-686,292-producing" strain of Actinoolanacete so.. and particularlypreferred is the ATCC No. 53771 strain.

In general, L-686,292 can be produced by culturing (fermenting) theabove-described "L-686,292-producing strain" in the presence ofL-683,590 in an aqueous nutrient medium containing sources ofassimilable carbon and nitrogen, preferably under submerged aerobicconditions (e.g. shaking culture, submerged culture, etc.). The aqueousmedium is preferably maintained at a pH of about 7 at the initiation andtermination (harvest) of the fermentation process. A higher pH leads tosubstantial and/or total loss of product. The desired pH may bemaintained by the use of a buffer such as morpholinoethanesulfonic acid(MES), morpholinopropanesulfonic acid (MOPS), and the like, or by choiceof nutrient materials which inherently possess buffering properties,such as production media described hereinbelow.

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, xylose, galactose, glycerin, starch, dextrin, and thelike. Other sources which may be included are maltose, rhamnose,raffinose, arabinose, mannose, salicin, sodium succinate, and the like.

The preferred sources of nitrogen are yeast extract, meat extract,peptone, gluten meal, cottonseed meal, soybean meal and other veqetablemeals (partially or totally defatted), casein hydrolysates, soybeanhydrolysates and yeast hydrolysates, corn steep liquor, dried yeast,wheat germ, feather meal, peanut powder, distiller's solubles, etc., aswell as inorganic and organic. nitrogen compounds such as ammonium salts(e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.),urea, amino acids, and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form, because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients, are also suitable for use. Whendesired, there may be added to the medium mineral salts such as sodiumor calcium carbonate, sodium or potassium phosphate, sodium or potassiumchloride, sodium or potassium iodide, magnesium salts, copper salts,cobalt salts, and the like. If necessary, especially when the culturemedium foams seriously, a defoaming agent, such as liquid paraffin,fatty oil, plant oil, mineral oil or silicone may be added.

The L-683,590 starting material can be obtained by the fermentation ofS. hygroscopicus var. ascomyceticus, ATCC No. 14891, as described inU.S. Pat. No. 3,244,592, and by the fermentation of S. hygroscopicussubsp. yakushimaensis No. 7278, (to produce FR-900520, or "FK-520",which is identical to L-683,590) as described in EPO Publication No.0184162 to Fujisawa, said above references hereby incorporated byreference for this particular purpose.

As to the conditions for the production of L-686,292 in massive amounts,submerged aerobic cultural conditions are preferred therefor. For theproduction in small amounts, a shaking or surface culture in a flask orbottle is employed. Furthermore, when the growth is carried out in largetanks, it is preferable to use the vegetative form of the organism forinoculation in the production tanks in order to avoid growth lag in theprocess of production of L-686,292. Accordingly, it is desirable firstto produce a vegetative inoculum of the organism by inoculating arelatively small quantity of culture medium with spores or mycelia ofthe organism produced in a "slant" and culturing said inoculated medium,also called the "seed medium", and then to transfer the culturedvegetative inoculum aseptically to large tanks. The fermentation medium,in which the inoculum is produced, is substantially the same as ordifferent from the medium utilized for the production of L-686,292 andis generally autoclaved to sterilize the medium prior to inoculation.The pH of the medium is generally adjusted to about 7.0 prior to theautoclaving step by suitable addition of an acid or base, preferably inthe form of a buffering solution.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

The fermentation is usually conducted at a temperature between about 20°C. and 40° C., preferably 25°-35° C., for a period of about 10 hours to24 hours, which may be varied according to fermentation conditions andscales. Preferably, the production cultures are incubated for about 24hours at 27° C. on a rotary shaker operating a 220 rpm, wherein the pHof the fermentation medium is maintained at 7.0 to harvest.

Preferred culturing/production media for carrying out the fermentationinclude the following media:

    ______________________________________                                                          g/l                                                         ______________________________________                                        Seed Medium A                                                                 Dextrose            1.0                                                       Dextrin             10.0                                                      Beef Extract        3.0                                                       Ardamine PH         5.0                                                       NZ Amine Type E     5.0                                                       MgSO.sub.4.7H.sub.2 O                                                                              0.05                                                     K.sub.2 HPO.sub.4    0.37                                                     Adjust pH to 7.1                                                              Add CaCO.sub.3 0.5 g/l                                                        Transformation Medium B                                                       Glucose             10                                                        Hycase SF           2                                                         Beef Extract        1                                                         Corn Steep Liquor   3                                                         Adjust PH to 7.0                                                              ______________________________________                                    

The produced L-686,292 can be recovered from the culture medium byconventional means which are commonly used for the recovery of otherknown biologically active substances. The L-686,292 substance producedis found int he cultured mycelium and filtrate, and accordingly can beisolated and purified from the mycelium and the filtrate, which areobtained by filtering or centrifuging the cultured broth, by aconventional method such as concentration under reduced pressure,lyophilization, extraction with a conventional solvent, such as methanoland the like, pH adjustment, treatment with a conventional resin (e.g.anion or cation exchange resin, non-ionic adsorption resin, etc.),treatment with a conventional adsorbent (e.g. activated charcoal,silicic acid, silica gel, cellulose, alumina, etc.), crystallization,recrystallization, and the like. A preferred method is solventextraction, particularly using methanol.

The product L-686,292 from the fermentation exhibits L-679,934antagonist activity as indicated by the "T-cell proliferation assay" andpossesses utility on this basis and exhibits the following physicalcharacteristics:

1. White amorphous powder

2. Solubility in methanol

3. Molecular weight of 763, as determined by electron impact massspectroscopy and is consistent with the assigned molecular structure inFIG. 1.

The L-686,292 obtained according to the fermentation processes asexplained above can be isolated and purified in a conventional manner,for example, extraction, precipitation, fractional crystallization,recrystallization, chromatography, and the like.

Suitable formulations of the material may also include conventionalpharmaceutically acceptable biolabile esters of L-686,292, formed viathe hydroxy groups on the molecule, such as the acetate.

It is to be noted that in the aforementioned fermentation reactions andthe post-treatment of the fermentation mixture therein, the tautomericand conformational isomer(s) of L-686,292, including those due torearrangement of the L-686,292 hemiketal ring system are also includedwithin the scope of the present invention.

The L-686,292 of the present invention possesses L-679,934 antagonistactivity and therefore is useful diagnostically as a tool to determinethe presence of FK-506 macrolide type immunosuppressants in naturalproduct broths, distinguishing such immunosuppressants from otherchemical families such as the cyclosporins. Diagnostic devices utilizingthis principle will be obvious to one skilled in the art.

The following examples are given for the purpose of illustrating thepresent invention and should not be construed as being limitations onthe scope or spirit of the instant invention.

EXAMPLE 1 Microorganism and Culture Conditions

Frozen vegetative mycelium (MA 6559) ATCC No. 53771 was thawed and usedto inoculate a 250 ml baffled shake flask containing 50 ml of autoclaved(sterilized) seed medium A consisting of (in units of grams/liter)dextrin 10.0, dextrose 1.0, beef extract 3.0, ardamine PH (YeastProducts, Inc.) 5.0, N-Z Amine type E 5.0, MgSO₄.7H₂ O 0.05, KH₂ PO₄0.37, and CaCO₃ 0.5. The PH of the seed medium was adjusted to 7.1before autoclaving. The seed was incubated in the seed medium at 27° C.for 24 hours on a rotary shaker operating at 220 rpm. A 2.5 ml aliquotof the resulting seed medium was used to inoculate a 250 ml non-baffledshake flask containing 50 ml of the following previously autoclaved(sterilized) transformation medium B. L-683,590 was added as a solutionin dimethylsulfoxide to achieve a final concentration of 0.1 mg/mlconcentration. The shake flask contents were subsequently incubated for24 hours at 27° C. on a rotary shaker operating at 220 rpm.

1. Transformation medium B consisted of (in grams/liter) glucose 10.0;Hycase SF 2.0; beef extract 1.0; corn steep liquor 3.0; where the pH wasadjusted to 7.0 before autoclaving.

Isolation and Purification Procedure for the Broth

The whole broth (500 ml) of transformation media B was extracted threetimes with methylene chloride (3×500 ml). Methylene chloride extractswere combined, dried over sodium sulfate, and concentrated under vacuumto an oily residue. The residue was dissolved in acetonitrile andsubjected to high performance liquid chromatography (HPLC) purification.

HPLC was carried out on Whatman Partisil 10 ODS-3, 9.4 mm×25 cm columnand monitored at 205 nm and 225 nm at 60° C. The column was developed atml./min with linear gradient from 0.1% aqueous H₃ PO₄ -CH₃ CN, 55:45 to0.1% aqueous H₃ PO₄ -CH₃ CN, 20:80 in 60 minutes. The compound wascollected during repeated injections of the above described extract. Thefractions at retention time 5 minutes were pooled, adjusted to pH 6.5and evaporated to remove acetonitrile. The compound was further purifiedusing a C₁₈ Sep-Pak (Waters Associates) and acetonitrile-water elutionsolvent to yield 1.2 mg. of product, designated as L-686,292.

Characterization

L-686,292 was characterized via NMR spectrometry yielding the proton NMRspectrum of FIG. 1, which confirms the assigned molecular structure.

EXAMPLE 2 T-Cell Proliferation Assay 1. Sample Preparation

Purified L-686.,292, as prepared by HPLC above, was dissolved inabsolute ethanol at 1 mg/ml.

2. Assay

Spleens from C57B1/6 mice were taken under sterile conditions and gentlydissociated in ice-cold RPMI 1640 culture medium (GIBCO, Grand Island,N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO).Cells were pelleted by centrifugation at 1500 rpm for 8 minutes.Contaminating red cells were removed by treating the pellet withammonium chloride lysing buffer (GIBCO) for 2 minutes at 4° C. Coldmedium was added and cells were again centrifuged at 1500 rpm for 8minutes. T lymphocytes were then isolated by separation of the cellsuspension on nylon wool columns as follows: Nylon wool columns wereprepared by packing approximately 4 grams of washed and dried nylon woolinto 20 ml plastic syringes. The columns were sterilized by autoclavingat 250° F. for 30 minutes. Nylon wool columns were wetted with warm (37°C.) culture medium and rinsed with the same medium. Washed spleen cellsresuspended in warm medium were slowly applied to the nylon wool. Thecolumns were then incubated in an upright position at 37° C. for 1 hour.Non-adherent T lymphocytes were eluted from the columns with warmculture medium and the cell suspensions were spun as above.

Purified T lymphocytes were resuspended at 2.5×10⁵ cells/ml in completeculture medium composed of RPMI 1640 medium with 10% heat-inactivatedfetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 2×10⁻⁵ M2-mercaptoethanol and 50 μg/ml gentamycin. Ionomycin was added at 250ng/ml and PMA at 10 ng/ml. The cell suspension was immediatelydistributed into 96 well flat-bottom microculture plates (Costar) at 200μl/well. Cultures containing ionomycin plus PMA were incubated with andwithout 1 ng/ml L-679,934 (FK-506) and various below-indicated dilutionsof the sample (above-described purified L-686,292). L-686,292 was addedin duplicate wells at 20 μl/well. The culture plates were then incubatedat 37° C. in a humidified atmosphere of 5% CO₂ -95% air for 44 hours.The proliferation of T lymphocytes was assessed by measurement oftritiated thymidine incorporation. After 44 hours of culturing, thecells were 1 pulse-labelled with 2 μCi/well of tritiated thymidine (NEN,Cambridge, Mass.). After another 4 hours of incubation, cultures wereharvested on glass fiber filters using a multiple sample harvester.Radioactivity of filter discs corresponding to individual wells wasmeasured by standard liquid scintillation counting methods(Betacounter). Mean counts per minute of replicate wells were calculatedand the results expressed as percent inhibition of tritiated thymidineuptake (proliferation) as follows: ##EQU1##

The results of % inhibition at various concentrations of L-686,292, inthe presence and absence of 1 ng/ml L-679,934, are presented in thefollowing table:

                  TABLE 1                                                         ______________________________________                                        Reversal of L-679,934 (FK-506)-Mediated                                       Inhibition of T-Cell Proliferation by L-686,292                                         % Inhibition                                                                                  L-686,292 &                                         L-686,292 (ng/ml)                                                                         L-686,292 only                                                                              L-679,934 (1 ng/ml)                                 ______________________________________                                         0          0             99                                                   31         0             99                                                   63         0             99                                                  125         0             96                                                  250         0             65                                                  500         0              3                                                  1000        0              0                                                  ______________________________________                                         Notes:                                                                        1. Mouse T cell cultures were pulsed with .sup.3 Hthymidine for 4 hours       prior to harvesting at 48 hours.                                              2. Standard L679,934 (1 ng/ml) gave 99% inhibition. The IC.sub.50 for         L679,934-mediated inhibition was 0.32 ng/ml (0.4 nM).                         3. IC.sub.50 of reversal = 250 ng/ml = 328 nM, for L686,292, and generall     in the range of 100 to 400 × 10.sup.-9 molar.                           4. Inhibition of proliferation by L679,934 was reversed by the addition o     50 units/ml of recombinant human IL2.                                    

                  TABLE 2                                                         ______________________________________                                        L-686,292 Reverses Inhibition of T-cell                                       Proliferation Mediated by L-679,934                                           but not by Cyclosporin A                                                      L-686,292 (ng/ml)                                                                         Inhibitor        % Inhibition                                     ______________________________________                                          0         L-679,934 (1 ng/ml)                                                                            97.1                                             1000        L-679,934 (1 ng/ml)                                                                             4.7                                              125        L-679,934 (1 ng/ml)                                                                            0                                                   62.5     L-679,934 (1 ng/ml)                                                                            65.7                                                31.3     L-679,934 (1 ng/ml)                                                                            93.4                                               0         Cyclosporin A (120 ng/ml)                                                                      86.6                                             1000        Cyclosporin A (120 ng/ml)                                                                      95.6                                             ______________________________________                                         Notes:                                                                        1. Inhibitors and L686,292 were added at the initiation of culture.           Cultures were pulsed with .sup.3 Hthymidine for 4 hours prior to              harvesting at 48 hours.                                                  

Based on the data above, it is seen that L-b 686,292 can be useddiagnostically as a tool to determine the presence of FK-506 macrolidetype immunosuppressants in natural product broths, distinguishing suchimmunosuppressants from other chemical families such as thecyclosporins.

What is claimed is:
 1. A compound which exhibits: a proton nuclearmagnetic spectrogram and molecular structure as depicted in FIG. 1, anda molecular weight of 763 as determined by electron impact massspectroscopy.